As used herein, the term "sterol" refers to derivatives of a fused, reduced ring system, cyclopenta[.alpha.] -phenanthrene, comprising three fused cyclohexane rings (A, B and C) in a phenanthrene arrangement, and a terminal cyclopentane ring (D) having the formula and carbon atom position numbering shown below: ##STR1## where R is an 8 to 10 carbon-atom sidechain.
Sterols are metabolically derived from acetate. Acetyl coenzyme A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to mevalonate in an irreversible reaction catalyzed by the enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). Through the sequential steps of isomerization, condensation and dehydrogenation, IPP is converted to geranyl pyrophosphate (GPP). GPP combines with IPP to form farnesyl pyrophosphate (FPP), two molecules of which are reductively condensed to form squalene, a 30-carbon precursor of sterols.
In yeast, squalene is converted to squalene epoxide, which is then cyclized to form lanosterol. Lanosterol has two methyl groups at position 4, a methyl group at position 14, a double bond at position 8(9) and an 8 carbon sidechain of the formula: EQU CH.sub.3 CH(CH.sub.2).sub.2 CH.dbd.C(CH.sub.3).sub.2.
Lanosterol is sequentially demethylated at positions 14 and 4 to form zymosterol (cholesta-8,24-dienol), which is converted to ergosterol (ergosta-5,7,22-trienol), the most abundant sterol of naturally occurring, wild-type yeast via a series of five enzymatic reactions schematically diagramed in FIG. 1.
The five reactions are:
a. methylation of the carbon at position 24, catalyzed by a 24-methyltransferase;
b. movement of the double bond at position 8(9) to position 7(8), catalyzed by a .DELTA.8.fwdarw..DELTA.7 isomerase;
c. introduction of a double bond at position 5(6), catalyzed by a 5-dehydrogenase (desaturase);
d. introduction of a double bond at position 22(23), catalyzed by a 22-dehydrogenase (desaturase); and
e. removal of a double bond at position 24(28), catalyzed by a 24(28)-hydrogenass (reductase).
In wild-type yeast of the species Saccharomyces cerevisiae (S. cerevisiae), the predominant order of these reactions is thought to be a, b, c, d and e. [Parks et al., CRC Critical Reviews in Microbioloy, 6:301-341 (1978)].
According to such a predominant pathway, zymosterol is converted sequentially to fecosterol [ergosta-8,24(28)-dienol], episterol [ergosta-7,24(28)-dienol], ergosta-5,7,24(28)-trienol, ergosta-5,7,22,24(28)-tetraenol, and finally ergosterol.
If the enzymes catalyzing the reactions involved in the predominant pathway are substrate specific, then one would expect to find only the six sterols set forth above in yeast. Such, however, is not the case. Eighteen sterols have been found and described. [See, e.g., Parks et al., CRC Critical Reviews in Microbioloy, 6:301-341 (1978); Woods et al., Microbios, 10(A):73-80 (1974); Bard et al., Lipids, 12:645-654 (1977) (See Table 1)]. Thus, at least some of the enzymes are not substrate specific.
TABLE 1 ______________________________________ Required* Sterol Enzymes ______________________________________ 1. Zymosterol (cholesta- none 8,24-dienol) 2. fecosterol (ergosta- a 8,24(28)-dienol) 3. episterol (ergosta- a,b 7,24(28)-dienol) 4. ergosta-5,7,24(28)- a,b,c trienol 5. ergosta-5,7,22, a,b,c,d 24(28)-tetraenol 6. ergosterol (ergosta- a,b,c,d,e 5,7,22-trienol) 7. ergosta-7,22,24 a,b,d (28)-trienol 8. cholesta-7,24- b dienol 9. cholesta-5,7,24- b,c trienol 10. cholesta-5,7,22,24- b,c,d tetraenol 11. ergosta-5,7-dienol a,b,c,e 12. ergosta-7,22-dienol a,b,d,e 13. ergosta-7-enol a,b,e 14. ergosta-5,8-dienol a,c,e 15. ergosta-5,8,22- a,c,d,e trienol 16. ergosta-8,22-dienol a,d,e 17. ergosta-8-enol a,e 18. ergosta-8,14,24(28)- a trienol ______________________________________ *Enzymes theoretically required for the synthesis of the designated sterol.
Despite the lack of substrate specificity, one might expect that specific alterations in the sterol biosynthetic pathway would have predictable consequences. Currently available data show that such predictability is not present.
For example, mutant S. cerevisiae with a defect in the expression of zymosterol-24-methyl-transferase (enzyme a), which mutants are designated erg6, might be expected to accumulate sterols 1 and 8-10 of Table 1, which sterols theoretically do not require the action of enzyme a for their synthesis. Parks et al., CRC Critical Reviews in Microbiology, 6:301-341 (1978), however, report that erg6 mutants accumulate only zymosterol (#1), cholesta-5,7,24-trienol (#9) and cholesta-5,7,22,24-tetranol (#10). Bard, M. et al., Lipids, 12:645-654 (1977), on the other hand, report that erg6 mutants accumulate only sterols #1 and #10.
Mutant S. cerevisiae with a defect in the expression of ergosta-5,7,24(28)-trienol-22-dehydrogenase (enzyme d), designated erg5, might be expected to accumulate sterols 1-4, 6, 8, 9, 11, 13, 14, 17 and 18. Parks et al., CRC Critical Reviews in Microbiology, 6:301-341 (1978) report, that erg5 mutants accumulate only ergosta-5,7-dienol (#11), ergosta-5,7,24(28)-trienol (#4), ergosta-8,14,24(28)-trienol (#18) and episterol (#3). In contrast, Bard et al., Lipids, 12:645-654 (1977) report that erg5 mutants accumulate zymosterol (#1), ergosta-5,7-dienol (#11), ergosta-5,7,24(28)-trienol (#4), ergosta-7,24(28)-dienol (#3) and ergosta-8,14,24(28)-trienol (#18).
Still further, mutant S. cerevisiae with a defect in episterol-5-dehydrogenase (enzyme c), designated erg3, might be expected to accumulate sterols 1-3, 7, 8, 12, 13 and 16-18. Parks et al., CRC Critical Reviews in Microbiology, 6:301-341 (1978) report that erg3 mutants accumulate only ergosta-7,22-dienol (#12), ergosta-8,22-dienol (#16), ergosta-7,22,24(28)-trienol (#7), fecosterol (#2) and episterol (#3).
These data, taken together, show that specific defects in the expression of one sterol synthetic enzyme do not lead to predictable changes in sterol accumulation. A similar degree of unpredictability is found when sterol accumulation is examined in mutants having two defects in enzymes of the sterol biosynthetic pathway.
Thus, for example, erg5-erg6 double mutants (defects in enzymes d and a) might be expected to accumulate sterols 1, 8 and 9. Parks et al. and Bard et al., above, report that erg5-erg6 double mutants accumulate only zymosterol (#1) and cholesta-5,7,24-trienol (#9).
These data relating to sterol accumulation in yeast show that specific alterations in enzyme activity do not result in predictable changes in sterol accumulation. The data further show a lack of agreement between different investigators studying identical alterations. The present invention furnishes a solution to the problem of unpredictability by providing a method and composition for increasing the accumulation of squalene and specific sterols in yeast.